1. Field of the Invention
The present invention in the fields of microbiology, immunology and medicine is directed to vaccine compositions and methods for preventing or treating mycobacterial diseases that are particularly suited to immunize a subject in mid-life who has been vaccinated neonatally or in early childhood with BCG and in whom protective immunity has waned. Thirty proteins from Mycobacterium tuberculosis are disclosed as having the desired immunogenicity and T cell stimulatory activity for such use. A preferred protein is Ag85A, the secreted product (SEQ ID NO:31) of the Rv3084c gene.
2. Description of the Background Art
Current epidemiologic data indicates that disease caused by the facultative intracellular bacterial parasite Mycobacterium tuberculosis (M. tuberculosis or “Mtb”) remains a serious global problem with around 8 million new cases per year, and with recent disturbing evidence that the death rate may be increasing (B. R. Bloom et al., Science 257:1055-64 (1992); M. C. Raviglione et al., Lancet 350:624-9 (1997); P. J. Dolin et al., Bull World Health Organ 72, 213-20 (1994); D. E. Snider, Jr., et al., N Engl J Med 338:1689-90 (1998).
For several decades the M. bovis-derived bacillus Calmette Guerin (BCG) has been the only widely used vaccine for tuberculosis (“TB”). BCG organisms, a strain of M. bovis, has been the only widely used vaccine for TB. However, accumulating data from clinical trials and subsequent meta-analysis (G. A. Colditz, et al., JAMA 271:698-702 (1994); J. A. Sterne et al., Int J Tuberc Lung Dis 2:200-7 (1998)) are revealing its general ineffectiveness in adults. It is noteworthy that adults vaccinated with BCG as young children become relatively unprotected. As a result many investigators are seeking to develop a new TB vaccine (I. M. Orme, Adv Vet Med 41:135-43 (1999); 1. M. Orme, Infect Dis Clin North Am 13:169-85, vii-viii (1999)); virtually all efforts are directed towards discovering new candidate vaccines that can be used in either or both prophylactic or immunotherapeutic modes (P. Andersen, Scand J Immunol 45:115-31 (1997). M. A. Horwitz et al., Proc Natl Acad Sci USA 92, 1530-4 (1995); D. B. Lowrie, et al., Nature 400:269-71 (1999)).
The mechanism underlying the gradual loss of effectiveness of BCG as the neonatally inoculated subject reaches 10-15 years of age is poorly understood. One possibility is that immunological memory generated by BCG has disappeared so that the subject is now the functional equivalent of a naive host that should be vaccinated with a new candidate vaccine that is designed to induce primary immunity as does BCG. An alternate possibility is slowly declining immunological memory can be re-induced by boosting with a candidate antigen that is specifically recognized by host memory cells (primarily T cells). The present invention is directed to the latter possibility and indeed discloses a number of Mtb antigens that can restimulate T cell memory and protective immunity.
The recent elucidation of the entire genome sequence of M. tuberculosis strain H37Rv (Cole, S. T. et al., Nature 1998, 393:537-544) is one of the most significant advancements to occur in tuberculosis research over the last decade and this data is now the backbone for several facets of tuberculosis research, including detailed analyses of the proteome of M. tuberculosis. Several laboratories have published two-dimensional (“2-D”) protein maps of the subcellular-fractions of M. tuberculosis (Jungblut, P R. et al., Mol. Microbiol. 1999, 33:1103-1117; Sonnenberg, M G and Belisle, J T, Infect. Immun. 1997, 65:4515-4524). Others have applied this technology to evaluate the proteins produced by Mycobacterium spp. during intracellular growth (Sturgill-Koszycki, S et al. Electrophoresis 1997, 18:2558-2565; Bai-Yu, L. et al., J. Clin. Invest. 1995, 96: 245-2496). Proteomics is also a facile approach to identifying immunodominant molecules. Through the use of 2-D PAGE and Western blot analysis, Samanich et al., J. Infect. Dis. 1998, 178, 1534-1538, recently defined 26 proteins of M. tuberculosis that reacted with antibodies of tuberculosis patients, and three of theses were determined to have potent serodiagnostic potential. See also, Laal et al., U.S. Pat. No. 6,245,331. However such an approach has not been applied on a large scale to the molecular identification of the T cell antigens of M. tuberculosis. 
T cells mediate the protective immune response to an M. tuberculosis infection (Orme, I M et al., J. Infect. Dis. 1993, 167:1481-1497), and elucidation of the T cell antigens has been a driving force in the identification of M. tuberculosis proteins. However, many of the proteins assessed for T cell reactivity were selected because they were abundant, easily purified or reactive to existing monoclonal antibodies (mAbs). Realizing that the study of T cell antigens required a systematic approach, Andersen and Heron, J. Immun. Methods 1993. 16, 29-39, developed a whole-gel elutor to systematically fractionate short-term M. tuberculosis culture filtrate proteins (CFPs) by size. This group and others have used this technique to identify T cell antigens and develop 1-D patterns of proteins inducing T cell reactivity (Boesen, H et al., Infect. Immun. 1995, 63:1491-1497; Roberts, A D et al., Immunol. 1995, 85:502-508). Andersen's group (Weldingh, K et al., FEMS Immunol. Med. Microbiol. 1999, 23:159-164) recently coupled this technique with preparative IEF to provide greater resolution. However, this work was focused on characterization of a small number of proteins. Similarly, Gulle et al., Vet. Immunol. Immunopath. 1995, 48:183-190) performed T cell proliferative studies of M. bovis BCG proteins that were electroeluted from 2-D polyacrylamide gels. Although this work revealed a detailed 2-D pattern of potential T cell antigens, those proteins found to be immunogenic were not identified. Moreover this approach had several inherent limitations such as, the amount of protein obtained by elution from a single gel was insufficient for multiple analyses, and the concentration of individual proteins tested varied.
Several laboratories, including the present inventors', have shown that proteins found in Mtb culture filtrates are highly immunogenic and have promise as candidate vaccines (Orme, supra). One member of this pool, disclosed herein to be a preferred boosting antigen, is the mycolyl transferase A enzyme also termed Ag85A (J. T. Belisle, et al., Science 276:1420-2 (1997)) (and also known as FbpA, encoded by the Mtb gene Rv3804c (available in GenBank).
The present inventors and their colleagues demonstrated that the majority of CD4+ T cells accumulating in the lungs of immune mice after challenge infection recognize this antigen (A. M. Cooper et al., Tuber Lung Dis 78:67-73 (1997)).
Mice infected with M. tuberculosis generate T cells that recognize the Ag85 protein. As a consequence, investigators have attempted to use Ag85 protein as a primary vaccine. It appears that no one has shown any vaccine or antigen preparation that approaches the protection conferred by the “gold standard” vaccine, BCG.
Horwitz MA et al., 1995, supra, claimed that Ag85 protein protected guinea pigs against aerosol TB. This study was said by the authors to demonstrate that immunization with the Mtb 30-kDa major secretory protein (Ag85A), alone or in combination with other abundant extracellular Mtb proteins induced strong cell-mediated immune responses and substantial protective immunity against aerosal challenge with virulent Mtb bacilli in the highly susceptible guinea pig model of pulmonary tuberculosis. Protection was manifested by decreased morbidity (including decreased weight loss and mortality), and decreased growth of Mtb in the lungs and spleens compared with sham-immunized controls. The authors concluded that purified major extracellular proteins of Mtb are candidate components of a subunit vaccine against TB. It is noteworthy that Ag85 was given mixed with other proteins, and an adjuvant that cannot be used in humans. Moreover, the experiment had no positive control, it was halted before the negative control animals died. This paper has been widely criticized for its lack od proper scientific methodology.
In contrast, the present inventors conducted a controlled, uncontaminated test of Ag85 as a primary vaccine in guinea pigs and found no reduction in lung bacterial load.
U.S. Pat. No. 5,736,524 discloses the use of primary DNA vaccines that encode Ag85. However, experience since this document became public has shown that the vaccines disclosed therein are even less effective than BCG.
Moreover, booster inoculation of BCG has proven ineffective. Thus, people who were neonatally vaccinated with BCG are at risk for TB as their T cell reactivity and antibody titers decline over time after primary vaccination.
While there is a desperate need to develop new TB vaccines to deal with the global emergency in general, in more advanced countries TB continues to be relatively more common in the elderly (W. W. Stead et al., Annu Rev Med 42: 267-726 (1991); W. W. Stead, Int J Tuberc Lung Dis 2: S64-70 (1998)). In the United States, for instance, people over the age of 65 have for some time now represented the fastest growing segment of the overall population (US Census Bureau. Worldwide Web page having the URL:.census.gov/socdemo/www/agebrief.html). While primary TB sometimes occurs in these individuals, the majority of cases are thought to be due to reactivation of latent disease acquired many decades earlier (Steadl, 1998, supra). h view of this, the vaccine strategy disclosed herein is applicable for preventing reactivation TB in the elderly.
Citation of the above documents is not intended as an admission that any of the foregoing is pertinent prior art. All statements as to the date or representation as to the contents of these documents is based on the information available to the applicant and does not constitute any admission as to the correctness of the dates or contents of these documents.